The polymerase chain reaction (PCR) is a widely used technique in biology. Conventional PCR instruments consist of plastic plates and hot plates. The plastic plates house multiple tubes that serve as reaction chambers and are placed on the hot plates for thermal cycling. PCR amplification is very slow due to the large sample volumes as well as the thick walls of the plastic tubes. Only a single protocol can be performed each time. Development of a new generation PCR has focused on rapid, multi-chamber, independent control PCR.
An important advantage of miniaturized PCR is fast speed. It has been shown that quick and accurate thermal cycling can be readily achieved, with very small sample volume consumption, by using micro-PCR techniques (1). Most of the recently developed micro-PCR equipment has been fabricated using microelectronic techniques which are in widespread use for the manufacture of integrated circuits (ICs) for semiconductor and micro-electromechanical (MEMS) systems. Reaction chamber, heat sources, and temperature sensors are integrated on a silicon substrate which has excellent thermal properties.
However, due to the high grade materials and sophisticated processing that are involved, micro-PCR equipment of this type tends to be expensive, making it unsuitable for use in environments, such as biomedical applications, in which a disposable chip is usually required.
Baier [2] has shown that a low cost multi-chamber thermal cycler, using the same thermal protocols possible in silicon, can be developed. Plastics have been investigated for use in disposable micro-PCR, but plastics have only fair thermal conductivity compared to silicon which might result in a slow thermal response and poor temperature uniformity.
An ultra thin-walled multi-well plate for thermal cycling has been described by Tretiakov and Saluz (3). The thin wall (30-50_m) reduced the thermal delay through the wall efficiently but use of a tube-like (V) shape for the chamber itself increased the thermal delay, thick samples being the dominant source of this delay. Another drawback of the tube approach is evaporative loss from the sample due to the presence of air in the tube. An example of this type of structure is schematically illustrated in FIGS. 1a and 1b where FIG. 1b is a cross-section of part of FIG. 1a. FIG. 1a shows an array of tube holders 12 distributed over the surface of heating block 11. As seen in FIG. 1b, each tube holder allows a disposable sample tube 13 to be kept in position close to heater 11.
In designs such as that shown in FIG. 1b, PCR speed is limited by the heat block ramping rate and the thermal diffusion delay time through the plastic wall as well as the sample itself. A similar approach has been described by Icke et al. (4). The air gap is miniaturized with an appropriate large thermal contact using a U shaped chamber. O'Keefe et al. disclose a multi-chamber plate where each chamber is fully filled by a sample without any air (5). However, said chamber is open on two sides, making full sealing for the reaction questionable. It is suitable for sample handling rather than for use as a reaction chamber. The preferred application of both the above designs is for a single protocol.
The present invention discloses a low cost disposable plastic chip that is well suited for use as a multiplexing thermal cycler.